Ya-Ling Hsu1, Po-Lin Kuo. 1. Cell Biology Laboratory, Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan.
Abstract
INTRODUCTION: The survival of osteoblasts is one of the determinants of the development of osteoporosis. This study is the first to investigate the osteoblastic differentiation induced by diosmetin, a flavonoid derivative, in osteoblastic cell lines MG-63, hFOB, and MC3T3-E1 and bone marrow stroma cell line M2-10B4. MATERIALS AND METHODS: Osteoblastic differentiation was determined by assaying alkaline phosphatase (ALP) activity and mineralization degree and measuring various osteoblast-related markers using ELISA. Expression and phosphorylation of Runt-related transcription factor 2 (Runx2), protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase (ERK), p38, and c-jun-N-terminal kinase (JNK) was assessed by immunoblot. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was assessed by EMSA. Genetic inhibition was performed by small hairpin RNA plasmids or small interfering RNA (siRNA) transfection. RESULTS: Diosmetin exhibited an effect on osteoblastic maturation and differentiation by means of ALP activity, osteocalcin, osteopontin, and type I collagen production, as well as Runx2 upregulation. Induction of differentiation by diosmetin was associated with increased PKCdelta phosphorylation and the activations of Rac1 and p38 and ERK1/2 kinases. Blocking PKCdelta by siRNA inhibition significantly decreased osteoblastic differentiation by inhibiting Rac1 activation and subsequently attenuating the phosphorylation of p38 and ERK1/2. In addition, blocking p38 and ERK1/2 by siRNA transfection also suppressed diosmetin-induced cell differentiation. CONCLUSIONS: In this study, we show that diosmetin induced osteoblastic differentiation through the PKCdelta-Rac1-MEK3/6-p38 and PKCdelta-Rac1-MEK1/2- ERK1/2-Runx2 pathways and that it is a promising agent for treating osteoporosis.
INTRODUCTION: The survival of osteoblasts is one of the determinants of the development of osteoporosis. This study is the first to investigate the osteoblastic differentiation induced by diosmetin, a flavonoid derivative, in osteoblastic cell lines MG-63, hFOB, and MC3T3-E1 and bone marrow stroma cell line M2-10B4. MATERIALS AND METHODS: Osteoblastic differentiation was determined by assaying alkaline phosphatase (ALP) activity and mineralization degree and measuring various osteoblast-related markers using ELISA. Expression and phosphorylation of Runt-related transcription factor 2 (Runx2), protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase (ERK), p38, and c-jun-N-terminal kinase (JNK) was assessed by immunoblot. Rac1 activity was determined by immunoprecipitation, and Runx2 activity was assessed by EMSA. Genetic inhibition was performed by small hairpin RNA plasmids or small interfering RNA (siRNA) transfection. RESULTS:Diosmetin exhibited an effect on osteoblastic maturation and differentiation by means of ALP activity, osteocalcin, osteopontin, and type I collagen production, as well as Runx2 upregulation. Induction of differentiation by diosmetin was associated with increased PKCdelta phosphorylation and the activations of Rac1 and p38 and ERK1/2 kinases. Blocking PKCdelta by siRNA inhibition significantly decreased osteoblastic differentiation by inhibiting Rac1 activation and subsequently attenuating the phosphorylation of p38 and ERK1/2. In addition, blocking p38 and ERK1/2 by siRNA transfection also suppressed diosmetin-induced cell differentiation. CONCLUSIONS: In this study, we show that diosmetin induced osteoblastic differentiation through the PKCdelta-Rac1-MEK3/6-p38 and PKCdelta-Rac1-MEK1/2- ERK1/2-Runx2 pathways and that it is a promising agent for treating osteoporosis.
Authors: Walaa G Hozayen; Mohamed A El-Desouky; Hanan A Soliman; Rasha R Ahmed; Amal K Khaliefa Journal: BMC Complement Altern Med Date: 2016-06-02 Impact factor: 3.659